The goals of this proposal are to determine the role of the indispensable branched-chain amino acids (BCAAs) in energy metabolism and nutrient partitioning. There is evidence that increasing dietary protein has a beneficial effect on insulin sensitivity, satiety, lean body mass, and resistance to obesity. Leucine is the primary protein-derived nutrient signal. Paradoxically, plasma BCAAs are elevated in obesity, and there is evidence for dysregulation of BCAA metabolism in the obese state. To determine the role of leucine versus its metabolites in nutrient signaling, a mouse with a global deletion of the first enzyme in the BCAA catabolic pathway, the mitochondrial branched-chain aminotransferase, was generated (BCATm KO). This animal has chronically elevated plasma and tissue BCAAs and does not exhibit the neurologic symptoms of Maple Syrup Urine Disease. The BCATm KO mouse has enhanced energy expenditure, is more insulin-sensitive, is lean, and is resistant to diet-induced obesity. Elevated energy expenditure in this animal is explained in part by a novel protein futile cycle (enhanced rates of protein synthesis and degradation). The concept that protein futile cycling contributes to enhanced energy expenditure is new and novel. In this proposal, the mechanisms underlying the increased muscle proteolysis will be investigated and the hypothesis that the reduced adiposity (lean phenotype) results from reduced lipogenesis and/or reduced adipogenesis in the BCATm KO will be tested as well. We will determine whether a new mouse that expresses BCATm in liver (BCATm LivTg) is an appropriate model for humans, because humans express BCAT in the liver. Finally, the discovery that the first two enzymes in BCAA catabolism associate to form a supramolecular complex (BCAA metabolon) that also contains enzymes that are key players in carbohydrate, fatty acid, and amino acid metabolism provides a new mechanism for cross-talk between the BCAAs and other nutrient pathways. This regulation is independent of known signaling pathways activated by leucine and insulin. The hypothesis that the BCAAs/leucine communicate with the other macronutrients through this metabolon will be tested at the molecular level. Metabolon function in vitro, in cultured cells and finally in mitochondria isolated from the transgenic animals with altered or blocked BCAA catabolism will be determined. Results from this study will provide new insights into the role(s) played by BCAAs and high protein diets in weight loss and energy expenditure.